Crystallization of chlortetracycline



Nov. 3, 1953 E. R. HARMS 2,658,077

CRYSTALLIZATION OF CHLORTETRCYCLINE Filed Aug. 20, 1949 4 Sheets-Sheet l f4 deca/5 5a/0770 of C/of/efracyche Nov. 3, 1953 E. R. HARMS 2,658,077

CRYSTALLIZATION OF CHLORTETRACYCLINE Filed Aug. 20, 1949 4 Sheets-Sheet 2 ATTO R N EY Nov. 3, 1953 E, R, HARMS 2,658,077

CRYSTALLIZATION oF CHLORTETRACYCLINE Filed Aug. 20, 1949 4 Sheets-Sheet 5 (prefer @Cefa/7) Td/067700 ATTORNEY Nov. 3, 1953 E. R. HARMs 2,658,077

CRYSTALLIZATION OF CHLORTETRACYCLINE Filed Aug. 20, 1949 4 Sheets-Sheet 4 www@ www f FW my Conf/'nue 05 /'/7 ATTORNEY Patented Nov. 3, 1953 CRYSTALLIZATION OF CHLORTETRA- CYCLIN E Edward R. Harms, Park Ridge, N. J., assignor to American Cyanamid Company, New York, N. Y., a corporation of Maine Application August 20, 1949, Serial No. 111,581

6 Claims.

This invention relates to a method for the isolation of chlortetracycline from the media from which it is produced, or a solution in which it is resent, and the purification of the chlortetracycline. It has for its object a distinctive and novel process whereby the chlortetracycline may be most conveniently recovered from an aqueous solution and has as a further object the transformation of the chlortetracycline to a therapeutically desirable form. The organism Streptomyces aureofacz'ens during its growth produces a substance known as chlortetracycline which has peculiar and remarkable activity as an antibiotic having a greater therapeutic range than any of the previously known antibiotics. The antibiotic and certain methods of fermentation, and certain uses therefor are set forth in an application of Benjamin M. Duggar, Serial Number 7,592, iled February 11, 1948, now Patent 2,432,055, September 13, 1949. Additional information on both the organism and the properties of chlortetracycline are disclosed in the Annals of the New York Academy of Sciences, volume 51, article 2, November 1948; and much of the current medical literature contains references to remarkable results being presently attained with this potent antibiotic.

In the accompanying drawings, Figure 1 shows diagrammatically the method of obtaining puried chlortetracycline from an acidified aqueous solution containing chlortetracycline,

Figure 2 shows the preparation of the acidified aqueous solution of chlortetracycline from a crude chlortetracycline.

Figure 3 shows the preparation of the acidied aqueous solution directly by acidifying a fermentation mash.

Figure 4 shows the preparation of an alkaline cake of chlortetracycline from the fermentation mash.

Figure 5 shows the preparation of the acidiiied aqueous solution by extracting the alkaline cake with solvent and an acid, followed by removal of the solvent.

Figure 6 shows the preparation of the acidied aqueous solution by extracting the alkaline cake with a solvent, while still alkaline, and removal of the solvent and acidification.

Figure 7 shows the preparation of the acidied aqueous solution by extraction of the alkaline cake with acid water.

Chlortetracycline is produced and sold under the commercial designation Aureomycin which, in certain countries, is a trade-mark of the American Cyanemid .COmPaPY-- .-.Chlffleira- 2 cycline has been found to be a naphthacene derivative and while having a structure which strongly suggests tautomeric forms, is believed to exist with the following probable structure:

H 9/10'11 12 0H H 87 6 W 43-ooNH2 A numbering system is shown for the molecule. One proper name for the compound is l-dimethylamino-4, 6-dioxo-l0-chloro 11 methyl- 2,4a,5,7,11 pentahydroxy 1,4,4a,6,11,11a,12,12a octahydronaphthacene 3 carboxamide. The generic term for the compound is chlortetracycline, sometimes spelled chlorotetracycline (Note: Journal of the American Chemical Society, 74: 4976 (1952)).

Whereas in the past chromatographic adsorption and other methods have been used for the isolation of chlortetracycline, including steps of salting out of chlortetracycline into solvents, etc., I have discovered that the chlortetracycline may be particularly conveniently separated from an acid aqueous media by the addition of a solvent which may or may not be miscible with the water which is added in sufficient quantity to induce crystallization of the chlortetracycline from the aqueous phase, but which is not added in suliicient quantity to dissolve or extract the chlortetracycline. Frequently n-butanol is used. Preferably a salt such as sodium chloride is added to the aqueous layer for the several purposes of assisting in causing the chlortetracycline to come out of the aqueous layer, to insure a better break between the water and the solvent, and to cause the chlortetracycline to come out as the particular acid salt which is desired, frequently the hydrochloride. As steps in the preparation of the aqueous chlortetracycline containing material, the chlortetracycline may be concentrated from a more dilute acid aqueous solution by vacuum distillation. Alternatively the chlortetra cycline containing material may be caused to go into an aqueous layer by extraction of the chlortetracycline from a more dilute solution or from solids, such as the lter cake, by the use of a suitable solvent such as acetone; which is conveniently wet and serves as a source of water for the residual aqueous chlortetracycline-con- Certain steps in the refining of chlortetracycline herein disclosed are claimed in a copending application of Edward E. Starbird and Charles Pidacks, Serial No. 364,182, entitled Chlortetracycline, Purification `and Alkaline Earth Salts, filed June 25, 1953, which is a continuation-in-part of applications Serial Numbers 62,722 and 62,766 led INovember 3o, 1948, now

abandoned in favor of said Serial Number- The aqueous chlortetracycline-containing material may be the liquor resulting from anyof several preceding steps but most conveniently is either the fermentation mash itself or the liquid extract obtained by filtering the fermentation mash in the presence of an alkaline material containing ions selected from the group consisting of calcium, barium, magnesium and strontium ions, at a pH of from 6, to 11, the best fractionation being from 7 to 9, separating the resulting solids containing an insoluble salt of chlortetracycline and extracting the solids with an acid and using this resultant acid extract. Of course, a repeated or counter-current extraction .of the solids tends to give an improved yield. The acid solution containing the chlortetracycline is most efciently handled at a pH of less than about 4. If the pH is much higher than 4, the solubility characteristics are not as favorable; and for normal operations, it is preferred that the `pH be not far below 1 A lower `pI-l is theoretically satisfactory but in plant processes the corrosion induced by such an acidic solution causes corrosion problems or requires the use of remarkably acid resistant vessels, with no resulting compensating economic advantage. It is most convenient to use a 'pH of in the neighborhood of 1 to 2, and a `pH of '1.5 `is normally preferred as a reasonable compromise,y economically, of all of the factors involved including corrosion, t-he cost of the acid, solubilities, and recovery, although with some equipment corrosion factors may necessitate the treatment at a pH of 3. A solvent extraction may be used, such as acetone, or other ketone, ethanol, or other alcohol, and the chlortetracycline transferred to an acid aqueous slution by evaporation of the solvent, or other suitable method.

'I'he aqueous solution then containing the chlortetracycline may be concentrated by vacuum distillation if not already sufficiently concentrated. Some decomposition results from a temperature in excess of about 50 C. With normal plant conditions, a temperature in the range of to 40 C. is prefer-red as a compromise between the cost of cooling the condenser and the losses at the 'higher temperatures. The water may even be removed from the frozen phase, after cooling below freezing, but such drastic conditions are not necessary. The `distillation of a solvent, if present, and the water from the aqueous phase is continued until a suitable concentration of chlortetracycline in the residual aqueous phase is obtained. The degree of concentration depends upon purities Vconcentration should be at aqueous interface,

and various other factors. soluble in the aqueous phase to the extent of about 10 to 15 milligrams per milliliter at a pH of 1.5 at room temperature. It is preferred that the concentration be continued until the concentration of chlortetracycline in the aqueous phase is from 3 to 30A milligrams per milliliter, depending upon the impurities present. The least 0.3 milligram per milliliter for reasonable separation when salt is added. With certain chlortetracycline mashes the preferred concentration may be obtained by concentrating the aqueous phase t0 approximately 25% or less of the original mash volume. Upon .cooling and agitation the chlortetracycline will not readily separate from the aqueous phase. It-may however, if permitted to stand., separate as a comparatively impure product in an amorphous state. If however, in accordance with this invention a small quantity of a solvent and a salt is added, the material will tend to separate in a comparatively pure crystalline form. Best results are obtained if the quantity of salt is such that the solution will be nearly saturated. For the purposes of my invention `I prefer the use of sodium chloride as the salt because therefrom results chlortetracycline as the hydrochloride, and sodium chloride is cheap.

Other salts may be used. Potassium chloride and ammonium chloride give a nicely crystallized chlortetracycline hydrochloride. With calcium and lithium -chlorides the chlortetracycline has more of a tendency to remain in the amorphous state or separate as an oil; but these too may be used. vThe salts of other acids such as sodium sulfate, ammonium sulfate, lithium sulfate, disodium phosphate, mono-potassium phosphate, etc. may be used but the products are more diflicult to crystallize; and from the ther- Vapeutic standpoint the medical profession appears to prefer the hydrochloride, and accordingly, chlorides are the salts which are normally to be desired. Any amount of salt, less than a completely saturated solution, improves the separation of the chlortetracycline hydrochloride. Usually the more salt, the better yields. Slightly less than saturated solutions are con- 'veniez-it, as they give good yields, and do not themselves tend to precipitate the salt itself. rI he solvent present has lseveral purposes. It tends to dissolve certain of the solvent soluble impurities and remove them from the crystals. It tends to cause the separation to occur more readily, particularly if it furnishes a solventand it assists in the formation of the desired crystalline modification of chlortetracycline. lSome several solvents may be Used.

Among the more useful solvents are the middle chain alcohols. The lower alcohols tend to be more soluble in water than is desirable. Methyl,

ethyl and isopropyl alcohols cause separation -and as such give -a more conveniently utilized modification of the invention. The alcohols above decanol tend to become more viscous and Chlortetracycline is l asado?? harder to handle. The alkxy eth'anols-.and other polyhydric alcohols. and their esters are normally also satisfactory. Butanol .is a preferred solvent because it is readily obtainable commercially, at a, comparatively low price, is not so soluble in water as to cause diiiiculties in phase separation, and does not dissolve a disproportionately large quantity of the chlortetracycline.

Just sufficient butanol to form a separate phase gives the improved crystallization effect but if about 5% of normal butanol is added the butanol dissolves a larger proportion of the impurities without dissolving an unreasonably large proportion of the chlortetracycline. If more than butanol is added the loss of product in the solvent usually becomes disproportionately great.

The extraction is conveniently carried out at room temperature and the resulting crystalline chlortetracycline filtered from the two liquid phases. It may be washed with a solvent, or several solvents, and dried. Ethanol and water are cheap and convenient; other alcohols or ketones etc. may be used. The product obtained under my preferred conditions is a crystalline chlortetracycline, the purity of which depends somewhat upon the starting material. If other than the preferred procedures are used, additional purification may become necessary. An additional recrystallization usually improves the purity even though it may not be necessary.

The following specic examples are intended to show certain embodiments of the invention but it is to be understood that it is impractical EXAMPLE 1V Direct acidification To 4.9 liters of fermented mash containing chlortetracycline was added 135 milliliters of 6 normal hydrochloric acid which lowered the pH to 1.45. The mixture was agitated to insure thorough mixing, and solution of all the chlortetracycline. To an aliquot of 3000 milliliters of the resulting solution was added 300 grams of the filter-aid known as Hy-Flo Supercel and the material filtered through paper, and then washed with sufficient acidulated water that 3000 milliliters of filtrate were obtained. The filtrate was placed in a vacuum still and distilled within the temperature range of to 30 C. under vacuum until a 550 milliliter concentrate was obtained. 50 milliliters were retained for assay samples and the remaining 500 milliliters had added thereto 25 milliliters of normal butyl alcohol and 125 grams of sodium chloride. The mixture was agitated at room temperature for one-half hour, then stored overnight at 2 C. The crystals were removed by a Buchner funnel, washed once with a small quantity of alcohol, then with distilled water, and the thus isolated crystals were vacuum dried. There wasobtained a yield of 4.61v grams analyzing 300 gammas per milligram bya color- 6. .ometric assay vvli'iclil is' agrecvery. of 37 %..from the total mash to the crystals. f

EXAlVIPLE 2 AlkaZi-acid-process To 30 liters of chlortetracycline mash resulting directly from the fermentation tanks, at a pH of 6.34 was added 125 milliliters of a 10% slurry of calcium hydroxide and 300 grams of the iilteraid known as Hy-Flo Supercel. The material was agitated at room temperature for 10 minutes and the resulting material at a pH of 8.8 was filtered, and the cake washed in 3 liters of water. To 6 liters of water Ywas added 250 milliliters of a 1 to 4 mixture of sulfuric acid and the acidified water heated to 50 C. and the lter cake prepared above added. The pH which was then approximately 4.0 was lowered to a pH of 1.35 by adding additional 1 to 4 sulfuric acid, approximately 250 milliliters being required. The mixture was agitated at a temperature of between 45 and 50 C'. for 10 minutes and ltered. The residual cake was additionally extracted by a second 6 liters of water lhaving added thereto sufficient sulfuric acid to keep the pH at 1.45, the cake being stirred for 10 minutes and the material reltered. The cake was discarded and the extracts found to measure 15.5 liters. An aliquot of 10 liters of the-cooled extracts were vacuum distilled in a glass and stainless steel still at a temperature between 30 and 36 C. to 2600 milliliters. Toithe thus prepared concentrate, now at a pI-I of ll, was added 150 milliliters of normal butanol and 780 grams of sodium chloride. I'he mixture was agitated for 30 minutes and held in a cold room between 2-5" C'. overnight. The crystals which formed were washed with ethanol, then twice with water, then again with ethanol until the washing alcohol was light in color; the vtotal volume of water washings being milliliters andthe total volume of alcohol washings being milliliters. There was obtained a crystal weight of 10.8 grams of chlortetracycline hydrochloride with a crystal potency of 970 gammas per milligram. This is a remarkably pure chlortetracycline hydrochloride to be thus simply obtained..

EXAMTLE 3 Alkali-acid, acetone process 30 liters of fermentation mash had added thereto l0 normal sodium hydroxide until the pI-I was approximately 9; 50 milliliters being required. 500 grams of Hy-Flo Supercel filter-aid were added and the solids separated by filtration. To the cake was added 4.5 liters of 90% acetone (10% water) and thereto added sufficient l to 4 sulfuric acid to lower the pH to 1.4; approximately 450 milliliters being required. To the mixture was added 700 grams of sodium chloride, the mixture agitated `forli? minutes, then filtered. '7 liters of filtrate were obtained. Thev cake vwas re-extracted with 4.5 liters of 90% acetone, 90 milliliters of sulfuric acid being required to keep the pH at 1.4, and 350 grams of sodium chloride beingadded. 'I'he slurry was agitated` for l5 minutes and ltered; 3.8 liters ofA filtrate being obtained. The cake was washed with 3 liters of acetone and blown dry. The pooled acetone extracts were vacuum concentrated to 4150 milliliters, all of the residual being water, and adjusted to a pH of 2.5; 33.5 milliliters of sulfuric acid being required. The material was filtered, and to .theclear filtrate ,were added 207milliliters of butanol and 1245 grams'of sodium chloride. The mixture was agitated for one hour. then filtered on a Buchner funnel, Washedwith alcohol, water, and again with alcohol. There was obtained a yield of 13.5 grams of chlortetracycline hydrochloride analyzingV 1000 gammas per milligram; this amounting to a recovery of 48% of the mash activity. The product as thus obtained is satisfactory for therapeutic purposes.

EXAMPLE 4 Alkaline acetone process 8,150 liters of fermented chlortetracycline mash was adjusted to a pH of 8.5 with 10 normal sodium hydroxide. The material was filtered, and the filtrate was discarded. The cake was washed with a small volume of Water, then slurried at room temperature with 1200 liters of acetone. The mixture was adjusted to a pH of 10 with 10 normal sodium hydroxide and after minutes agitation, 400 kilograms of sodium chloride was added, and the mixture stirred for an additional 15 minutes, and then ltered. The cake was again extracted with an additional 800 liters of acetone, the mixture being adjusted to a pH of 10.2 with 10 normal sodium hydroxide, and agi;4 tated for 15 minutes, and the acetone removed by ltration. The pooled extracts now measuring 1,498 liters, were concentrated by vacuum distillation at a temperature between 25 and 35 C. until all of the acetone was removed. 'I'he concentrate is about 10% by volume of the original volume of the acetone. The aqueous concentrate was adjustedtol a pH of 1.5 with 4 molar sulfuric acid, 5% ci' its volume of butanol added, the mixture stirred and permitted to standv overnight. The crystals were removed, washed with ethanol, water, and again with ethanol, and then dried'. EXAMPLE 5 Alkali acid process A fermented chlortetra'cycline mash was adjusted to a pH of 8.5 with a 10% slurry oiY cal'- -cium hydroxide andthe mash ltered. The filter cake was addedY to a volume of water equivalent to of the original mash volume at a temperature of 50 C. andenough hydrochloric acid added to lower the pH to between 3 and 4. After the cake was thoroughly slurried-the pH was adjusted to 1.5 and the slurry agitated for an additional 10 minutes. The slurry was filtered and the cake extracted a second time with a similar volume of water, the water extracts combined and concentrated at atemperature of between 30A and 40 C. to 25% of their original volume. To the concentrate was added 5% by volumeof normal butanol and 30% weight/volumesodium chloride added and stirred for 90 minutes. The crystals thereby formed were separated, washed with ethanol, water, and again with ethanol and vacuum dried. The chlortetracycline hydrochloride as obtained was satisfactory fortherapeutic purposes.

EXAMPLE 6.

A fermented chlortetracycline mash was adjusted to a pH of 8.5 with a10% slurry of cal= cium hydroxide, and the mash filtered. The lter cake was added to a volume of water equivalent to 20% of the original mashvolume and enough hydrochloric acid'` added' to lower the pH toV 4. The cake was thoroughly mixed and the slurry filtered. The extract was concentrated by vacuum distillation` at a. temperature of' noti more than 50 C. to approximately 20% -of its original volume. To this concentrate was added 5% by volume of normal butanolk and 30% by weight of sodium chloride. The mixture was thoroughly stirred andthe crystals thereby formed separated, washed withethanol, and vacuum dried. Chlortetracycline hydrochloride was thereby recovered.l

EXAMPLES '7-18 A fermented mash was adjusted to a pH of 8.5 with 10 normal sodium hydroxide and the mash Illtered. The lter cake was added to a volume of water equivalent to 20% of the original mash volume, and enough hydrochloric acid added to lower the pH to approximately 1.5, and the resulting mixture again ltered. The extract was concentrated by vacuum distillation at a temperature between 30 and 40 C. to 25% of its original volume. To the concentrate was added 30 parts by weight of sodium chloride per parts by volume of liquid. The solution was separated into a number of aliquot parts and 5% by volume of various solvents were added thereto. In each instance crystals of chlortetracycline hydrochloride separated and were recoverable on a Buchner funnel.

Example Appear- No. Solvent ance Result 7 Anhlydrous ethalphase Crystals.

X10 Methanol Do. lsopropanol.. Do. n-Butanol Crystals at interface. Tert. butanol. Do. Sec butanol.- Do. Amyl alcoholm. Do. Hexyl alcohol- Do. Octyl alcohol Do. Caprylalcohol. Do. Decano] Do.

2-ethyoxy ethanol. Crystals.

In each instance the crystals as initially separated were a medium dark brown but upon washing with ethanol,A water, and ethanol, became a lighter yellow color;

EXAMPLES 19-28 Example Forni of chlortetracycline No. Salt recovered 19 Sodium chloride Crystals-hydrochloride salt.

20 Ammonium chloride.-.. Do.

2l Lithium chloride... Oil-hydrochloride salt.

Potassium chloride.

Crystals-hydrochloride salt.

Calcium chloride-. Aamorphous-hydrochloride s t. Sodium sulfate Amorphous sulfate salt. Ammonium sulfate.. Do. Lithium sulfate Do. Dl-sodium hydrogen Amorphous phosphate salt.

phosphate. Potassium dl-hydrogcn Oily phosphate salt.

phosphate.

The chlortetracycline obtained in the oily or amorphous form could be separated and con'- verted to a crystalline hydrochloride by treating itwith` a small portion of anhydrous ethanol and concentrated hydrochloric acid.

9i l EXAMPLE 29 High solvent To an aliquot obtained as in the preceding series of examples was added 30 parts by Weight `lof sodium chloride to 100 parts by volume and 10% by volume of normal butanol. After standing in a chill chamber for 24 hours, the crystals were iiltered oiT. The larger volume of butanol idissolved more of the impurities so that the chlortetracycline hydrochloride obtained appeared to be in a slightly purer form; but the yield `Was lessened because of the amount dissolved by butanol.

EXAMPLE 30 One liter of fermentation mash was raised to a pH of approximately 9 by the addition of 10 normal sodium hydroxide, 25 grams of a diatomaceous earth ilter-aid Were added, and the solids separated by filtration. The cake, after Washing, was added to 250 milliliters of Water acidiiied to a pH of 1.35 With sulfuric acid, and after stirring, additional sulfuric acid Was added to keep the pH at 1.35. The slurry was filtered, and to the acidied extract was added 5% by Volume of butanol and 25% weight per volume of sodium chloride. The mixture was stirred and chilled, and the resulting crystals of chlortetracycline hydrochloride separated in a Buchner funnel, and washed With water. Crystalline chlortetracycline hydrochloride Was thereby recovered.

EXAMPLE 31 To 5 liters of fermented mash containing chlortetracycline Was added suiicient 6 normal hydrochloric acid to acidify the material to a pH of 1.4. The mixture was thoroughly agitated, then filtered. To the filtrate was added 5% by volume of normal butyl alcohol and 30% weight per volume of sodium chloride. The mixture was agitated at room temperature for one hour, then stored overnight at 2 C. The crystals Were removed by a Buchner funnel and Washed as in Example l. Thereby Was obtained crystalline chlortetracycline hydrochloride.

EXAMPLE 32 grams of an impure chlortetracycline hydrochloride were suspended in l liter of distilled Water, and thereto added sucient hydrochloric acid to lower the pH to 1.0. The mixture was warmed to about 50 C. and stirred for 30 minutes to insure complete solution of all of the chlortetracycline present. Insolubles were removed by filtration, 250 grams of sodium chloride were added in 50 milliliters of normal butanol. The mixture was cooled with stirring to 2 C. and permitted to stand overnight in a chill room. The crystals thereby formed were separated on a 'Buchner funnel and Washed with ethanol, Water,-

Aaspects thereof in detail, as my invention I claim:

course possible to operate at higher temperatures, greater concentrations or lesser concentrations, etc. with, of course, compensating variations in the yield.

Having thus described and set forth certain 1. A method of isolating chlortetracycline hydrochloride which comprises forming an acid solution ata pI-I of less than 4 containing from .3 to 30 milligrams of chlortetracycline per milliliter, adding 25 to 30% of sodium chloride and .not more than 10% normal butanol, and separating the thus formed chlortetracycline hydrochloride from the combined aqueous and butanol liquid present.

2. A method of isolating chlortetracycline as the hydrochloride salt which comprises forming an acid solution containing chlortetracycline at a pH of between approximately 1 and 2, adding thereto from 25 to 30 parts by Weight of sodium chloride per parts by volume of solution and approximately 5% of normal butanol, agitating and chilling the mixture and recovering the thus formed chlortetracycline hydrochloride salt from the combined aqueous and butanol liquid present.

3. A method of isolating chlortetracycline which comprises adding a salt of the group consisting of sodium chloride, ammonium chloride, lithium chloride, potassium chloride, calcium chloride, sodium sulfate, ammonium sulfate, lithium sulfate, di-sodium hydrogen phosphate and potassium di-hydrogen phosphate, and a solvent in a quantity insumcient to dissolve the chlortetracycline present from the group consisting of ethanol, methanol, isopropanol, nbutanol, tertiary butanol, secondary butanol, amyl alcohol, hexyl alcohol, octyl alcohol, capryl alcohol, decanol and 2-ethoxy ethanol to an aqueous solution containing chlortetracycline at a pH of less than 4 at a concentration of at least .3 milligram per milliliter and separating and recovering the thus formed acid salt of chlortetracycline from the combined aqueous and solvent liquid present.

4. A method of isolating chlortetracycline as an acid salt which comprises adding a salt from the group consisting of sodium chloride, ammonium chloride, lithium chloride, potassium chloride, calcium chloride, sodium sulfate, ammonium sulfate, lithium sulfate, di-sodium hydrogen phosphate and potassium di-hydrogen phosphate, and an organic solvent in a quantity insuicient to dissolve the chlortetracycline present from the group consisting of ethanol, methanol, isopropanol, n-butanol, tertiary butanol, secondary butanol, amyl alcohol, hexyl alcohol, octyl alcohol, capryl alcohol, decanol and 2-ethoxy ethanol to an aqueous solution containing chlortetracycline at a pH of less than 4 and at a concentration of from 3 to 30 milligrams per milliliter until the solution is substantially saturated with the salt and separating and recovering the thus formed acid salt of chlortetracycline from the combined aqueous and solvent liquid present.

5. A method of isolating chlortetracycline as the hydrochloride salt which comprises forming an acid solution containing at least about 3 milligrams per milliliter of chlortetracycline, adding thereto suflicient sodium chloride to substantially saturate the aqueous layer and approximately 5% by volume of normal butanol, and separating the thus formed chlortetracycline hydrochloride from the combined aqueous and butanol liquid present, all at a pH of less than about 4.

6. A method of isolating chlortetracycline as an acid salt Which-comprises adding .aninorganic water-soluble salt of Va -mineral acid and lan at least partially Water-soluble hydroxyla-ted organicsolvent in a quantity -insuicient to dissolve the Chlor-tetracycline present to an aqueoussolution 4containing chlortetracycline at a pH of less than 4 and a concentration of approximately 3 to 30 milligrams per milliliter, and separating and recoveringr the thus formed salt of chlortetracycline, with an acid, said acid being the acid derived from the anionic portion of said inorganic Water-soluble salt.

EDWARD R. HARMS 12 References Cited in .the f lle of this patent UNITED ,SfljATES PATENTS Number Name Date 2,481,763 vI .ineWeaJ/er Sept. 13, 1949 2,482,055 'Duggar Sept. 13, 1949 OTHER VREFERENCES 

6. A METHOD OF ISOLATING CHLORTETRACYCLINE AS AN ACID SALT WHICH COMPRISES ADDING IN INORGANIC WATER-SOLUBLE SALT OF A MINERAL ACID AND AN AT LEAST PARTIALLY WATER-SOLUBLE HYDROXYLATED ORGANIC SOLVENT IN A QUANTITY INSUFFICIENT TO DISSOLVE THE CHLORTETRACYCLINE PRESENT TO AN AQUEOUS SOLUTION CONTAINING CHLORTETRACYCLINE AT A PH OF LESS THAN 4 AND A CONCENTRATION OF APPROXIMATELY 3 TO 30 MILLIGRAMS PER MILLIMETER, AND SEPARATING AND RECOVERING THE THUS FORMED SALT OF CHLORTETRACYCLINE, WITH AN ACID, SAID ACID BEING THE ACID DERIVED FROM THE ANIONIC PORTION OF SAID INORGANIC WATER-SOLUBLE SALT. 